A wide variety of RNAs encode small open-reading-frame (smORF/sORF) peptides, but their functions are largely unknown. Here, we show that Drosophila polished-rice (pri) sORF peptides trigger proteasome-mediated protein processing, converting the Shavenbaby (Svb) transcription repressor into a shorter activator. A genome-wide RNA interference screen identifies an E2-E3 ubiquitin-conjugating complex, UbcD6-Ubr3, which targets Svb to the proteasome in a pri-dependent manner. Upon interaction with Ubr3, Pri peptides promote the binding of Ubr3 to Svb. Ubr3 can then ubiquitinate the Svb N terminus, which is degraded by the proteasome. The C-terminal domains protect Svb from complete degradation and ensure appropriate processing. Our data show that Pri peptides control selectivity of Ubr3 binding, which suggests that the family of sORF peptides may contain an extended repertoire of protein regulators.

The FlyRNAi database of the Drosophila RNAi Screening Center (DRSC) and Transgenic RNAi Project (TRiP) at Harvard Medical School and associated DRSC/TRiP Functional Genomics Resources website (http://fgr.hms.harvard.edu) serve as a reagent production tracking system, screen data repository, and portal to the community. Through this portal, we make available protocols, online tools, and other resources useful to researchers at all stages of high-throughput functional genomics screening, from assay design and reagent identification to data analysis and interpretation. In this update, we describe recent changes and additions to our website, database and suite of online tools. Recent changes reflect a shift in our focus from a single technology (RNAi) and model species (Drosophila) to the application of additional technologies (e.g. CRISPR) and support of integrated, cross-species approaches to uncovering gene function using functional genomics and other approaches.

Our understanding of the genetic mechanisms that underlie biological processes has relied extensively on loss-of-function (LOF) analyses. LOF methods target DNA, RNA or protein to reduce or to ablate gene function. By analysing the phenotypes that are caused by these perturbations the wild-type function of genes can be elucidated. Although all LOF methods reduce gene activity, the choice of approach (for example, mutagenesis, CRISPR-based gene editing, RNA interference, morpholinos or pharmacological inhibition) can have a major effect on phenotypic outcomes. Interpretation of the LOF phenotype must take into account the biological process that is targeted by each method. The practicality and efficiency of LOF methods also vary considerably between model systems. We describe parameters for choosing the optimal combination of method and system, and for interpreting phenotypes within the constraints of each method.

Heterochromatin is enriched for specific epigenetic factors including Heterochromatin Protein 1a (HP1a), and is essential for many organismal functions. To elucidate heterochromatin organization and regulation, we purified Drosophila melanogaster HP1a interactors, and performed a genome-wide RNAi screen to identify genes that impact HP1a levels or localization. The majority of the over four hundred putative HP1a interactors and regulators identified were previously unknown. We found that 13 of 16 tested candidates (83%) are required for gene silencing, providing a substantial increase in the number of identified components that impact heterochromatin properties. Surprisingly, image analysis revealed that although some HP1a interactors and regulators are broadly distributed within the heterochromatin domain, most localize to discrete subdomains that display dynamic localization patterns during the cell cycle. We conclude that heterochromatin composition and architecture is more spatially complex and dynamic than previously suggested, and propose that a network of subdomains regulates diverse heterochromatin functions.

FlyRNAi (http://www.flyrnai.org), the database and website of the Drosophila RNAi Screening Center (DRSC) at Harvard Medical School, serves a dual role, tracking both production of reagents for RNA interference (RNAi) screening in Drosophila cells and RNAi screen results. The database and website is used as a platform for community availability of protocols, tools, and other resources useful to researchers planning, conducting, analyzing or interpreting the results of Drosophila RNAi screens. Based on our own experience and user feedback, we have made several changes. Specifically, we have restructured the database to accommodate new types of reagents; added information about new RNAi libraries and other reagents; updated the user interface and website; and added new tools of use to the Drosophila community and others. Overall, the result is a more useful, flexible and comprehensive website and database.

Chromatin insulators of higher eukaryotes functionally divide the genome into active and inactive domains. Furthermore, insulators regulate enhancer/promoter communication, which is evident from the Drosophila bithorax locus in which a multitude of regulatory elements control segment specific gene activity. Centrosomal protein 190 (CP190) is targeted to insulators by CTCF or other insulator DNA-binding factors. Chromatin analyses revealed that insulators are characterized by open and nucleosome depleted regions. Here, we wanted to identify chromatin modification and remodelling factors required for an enhancer blocking function. We used the well-studied Fab-8 insulator of the bithorax locus to apply a genome-wide RNAi screen for factors that contribute to the enhancer blocking function of CTCF and CP190. Among 78 genes required for optimal Fab-8 mediated enhancer blocking, all four components of the NURF complex as well as several subunits of the dREAM complex were most evident. Mass spectrometric analyses of CTCF or CP190 bound proteins as well as immune precipitation confirmed NURF and dREAM binding. Both co-localise with most CP190 binding sites in the genome and chromatin immune precipitation showed that CP190 recruits NURF and dREAM. Nucleosome occupancy and histone H3 binding analyses revealed that CP190 mediated NURF binding results in nucleosomal depletion at CP190 binding sites. Thus, we conclude that CP190 binding to CTCF or to other DNA binding insulator factors mediates recruitment of NURF and dREAM. Furthermore, the enhancer blocking function of insulators is associated with nucleosomal depletion and requires NURF and dREAM.

Store-operated Ca2+ entry is mediated by Ca2+ release-activated Ca2+ (CRAC) channels following Ca2+ release from intracellular stores. We performed a genome-wide RNA interference (RNAi) screen in Drosophila cells to identify proteins that inhibit store-operated Ca2+ influx. A secondary patch-clamp screen identified CRACM1 and CRACM2 (CRAC modulators 1 and 2) as modulators of Drosophila CRAC currents. We characterized the human ortholog of CRACM1, a plasma membrane-resident protein encoded by gene FLJ14466. Although overexpression of CRACM1 did not affect CRAC currents, RNAi-mediated knockdown disrupted its activation. CRACM1 could be the CRAC channel itself, a subunit of it, or a component of the CRAC signaling machinery.

The specificity of RNAi pathways is determined by several classes of small RNAs, which include siRNAs, piRNAs, endo-siRNAs, and microRNAs (miRNAs). These small RNAs are invariably incorporated into large Argonaute (Ago)-containing effector complexes known as RNA-induced silencing complexes (RISCs), which they guide to silencing targets. Both genetic and biochemical strategies have yielded conserved molecular components of small RNA biogenesis and effector machineries. However, given the complexity of these pathways, there are likely to be additional components and regulators that remain to be uncovered. We have undertaken a comparative and comprehensive RNAi screen to identify genes that impact three major Ago-dependent small RNA pathways that operate in Drosophila S2 cells. We identify subsets of candidates that act positively or negatively in siRNA, endo-siRNA, and miRNA pathways. Our studies indicate that many components are shared among all three Argonaute-dependent silencing pathways, though each is also impacted by discrete sets of genes.

The DNA damage checkpoint, the first pathway known to be activated in response to DNA damage, is a mechanism by which the cell cycle is temporarily arrested to allow DNA repair. The checkpoint pathway transmits signals from the sites of DNA damage to the cell cycle machinery through the evolutionarily conserved ATM (ataxia telangiectasia mutated) and ATR (ATM- and Rad3-related) kinase cascades. We conducted a genome-wide RNAi (RNA interference) screen in Drosophila cells to identify previously unknown genes and pathways required for the G₂-M checkpoint induced by DNA double-strand breaks (DSBs). Our large-scale analysis provided a systems-level view of the G₂-M checkpoint and revealed the coordinated actions of particular classes of proteins, which include those involved in DNA repair, DNA replication, cell cycle control, chromatin regulation, and RNA processing. Further, from the screen and in vivo analysis, we identified previously unrecognized roles of two DNA damage response genes, mus101 and mus312. Our results suggest that the DNA replication preinitiation complex, which includes MUS101, and the MUS312-containing nuclease complexes, which are important for DSB repair, also function in the G₂-M checkpoint. Our results provide insight into the diverse mechanisms that link DNA damage and the checkpoint signaling pathway.

The androgen receptor (AR) is a mediator of both androgen-dependent and castration-resistant prostate cancers. Identification of cellular factors affecting AR transcriptional activity could in principle yield new targets that reduce AR activity and combat prostate cancer, yet a comprehensive analysis of the genes required for AR-dependent transcriptional activity has not been determined. Using an unbiased genetic approach that takes advantage of the evolutionary conservation of AR signaling, we have conducted a genome-wide RNAi screen in Drosophila cells for genes required for AR transcriptional activity and applied the results to human prostate cancer cells. We identified 45 AR-regulators, which include known pathway components and genes with functions not previously linked to AR regulation, such as HIPK2 (a protein kinase) and MED19 (a subunit of the Mediator complex). Depletion of HIPK2 and MED19 in human prostate cancer cells decreased AR target gene expression and, importantly, reduced the proliferation of androgen-dependent and castration-resistant prostate cancer cells. We also systematically analyzed additional Mediator subunits and uncovered a small subset of Mediator subunits that interpret AR signaling and affect AR-dependent transcription and prostate cancer cell proliferation. Importantly, targeting of HIPK2 by an FDA-approved kinase inhibitor phenocopied the effect of depletion by RNAi and reduced the growth of AR-positive, but not AR-negative, treatment-resistant prostate cancer cells. Thus, our screen has yielded new AR regulators including drugable targets that reduce the proliferation of castration-resistant prostate cancer cells.